Novel acyltranserases and methods of using

ABSTRACT

Provided herein are novel acyltransferases and methods of using such novel acyltransferases in making medium-chain fatty acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Application No. 61/917,586 filed Dec. 18, 2013. The entirety ofthe prior application is incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DE-SC0001295,2009-05988, and IOS 0701919 awarded by the U.S. Department of Energy,the U.S. Department of Agriculture, and the National Science Foundation,respectively. The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure generally relates to transgenic plants.

BACKGROUND

Plants in the genus, Cuphea (Lythracea), accumulate high levels ofmedium chain fatty acids (MCFAs) in their seeds. MCFAs are useful in thechemical industry in the production of detergents, lubricants andbiofuels. Camelina sativa is a member of the Brassicaceae family, andhas been found to be a sustainable source of oil for petroleum products.A high proportion of polyunsaturated fatty acids in Camelina oil,however, has limited its usefulness in the biofuel industry. Therefore,methods of engineering the oil properties in Camelina or otheroil-producing organisms are desirable.

SUMMARY

In one aspect, a method of producing triacylglycerols (TAGs) comprisingmedium-chain fatty acids (MCFAs) in an organism is provided. Such amethod typically includes introducing a transgene into the organism,wherein the transgene comprises at least one nucleic acid sequenceencoding an acyltransferase, wherein the at least one acyltransferaseexhibits a substrate specificity for saturated fatty acids, therebyproducing TAGs comprising MCFAs in the organism.

In some embodiments, at least 20% (at least 40%, at least 50%, etc.,etc.) of the TAGs comprising MCFAs have a C8:0 or a C10:0 at sn-2position. In some embodiments, the saturated fatty acids are selectedfrom the group consisting of C8:0 and C10:0.

In some embodiments, the at least one acyltransferase is alysophosphatidic acid acyltransferase (LPAT) or a diacylglycerolacyltransferase (DGAT). In some embodiments, the at least oneacyltransferase is a lysophosphatidic acid acyltransferase (LPAT) and adiacylglycerol acyltransferase (DGAT). In some embodiments, the nucleicacid sequence encoding the LPAT is selected from the group consisting ofa sequence having at least 95% sequence identity to SEQ ID NO:1 and asequence having at least 95% sequence identity to SEQ ID NO:3. In someembodiments, the nucleic acid sequence encoding the DGAT is selectedfrom the group consisting of a sequence having at least 95% sequenceidentity to SEQ ID NO:7 and a sequence having at least 95% sequenceidentity to SEQ ID NO:9. In some embodiments, the nucleic acid sequenceencoding the at least one acyltransferase is selected from the groupconsisting of a nucleic acid sequence having at least 95% sequenceidentity to SEQ ID NO:1, a nucleic acid sequence having at least 95%sequence identity to SEQ ID NO:3, a nucleic acid sequence having atleast 95% sequence identity to SEQ ID NO:7, and a nucleic acid sequencehaving at least 95% sequence identity to SEQ ID NO:9.

In some embodiments, the organism further comprises a nucleic acidsequence encoding a medium-chain fatty acid (MCFA)-specific thioesteraseFatB. In some embodiments, the nucleic acid sequence encoding theMCFA-specific thioesterase FatB is selected from the group consisting ofa nucleic acid sequence having at least 95% sequence identity to SEQ IDNO:11, a nucleic acid sequence having at least 95% sequence identity toSEQ ID NO:13, and a nucleic acid sequence having at least 95% sequenceidentity to SEQ ID NO:15.

In some embodiments, the organism is selected from the group consistingof a plant and a microbe. In some embodiments, the plant is Camelinasativa.

In some embodiments, the transgene comprises a promoter. In someembodiments, the promoter is a seed-specific promoter. In someembodiments, the at least one nucleic acid sequence encoding anacyltransferase is operably linked to a seed-specific promoter. In someembodiments, the medium-chain fatty acids are produced in the seed.

In some embodiments, the introducing step is performed usingAgrobacterium transformation, particle bombardment, or electroporationof protoplasts.

In another aspect, a method of producing triacylglycerols (TAGs)comprising medium-chain fatty acids (MCFAs) is provided. Such a methodtypically includes providing an organism comprising a transgene, whereinthe transgene comprises at least one nucleic acid sequence encoding anacyltransferase, wherein the at least one acyltransferase exhibits asubstrate specificity for saturated fatty acids; growing the organismunder appropriate conditions; and obtaining TAGs comprising MCGAs fromthe organism. In some embodiments, the TAGs are used in biofuel, jetfuel, detergents, and chemical feedstocks.

In still another aspect, a method of increasing the amount oftriacylglycerols (TAGs) comprising medium-chain fatty acids (MCFAs) inthe seed oil of a plant is provided. Such a method typically includesproviding a plant comprising a nucleic acid encoding a FatB polypeptide;introducing a heterologous nucleic acid molecule into the plantcomprising at least one nucleic acid sequence encoding anacyltransferase, wherein the at least one acyltransferase exhibits asubstrate specificity for saturated fatty acids, thereby increasing theamount of TAGs comprising MCFAs in the seed oil of the plant withoutsignificantly changing the total oil content in the seed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS Part A

FIG. 1 shows the phylogenic relationship in deduced amino acid sequencesof LPATs. Amino acid sequences of 6 LPATs from Cuphea pulcherrima (blacktriangle) and 1 LPAT from Cuphea viscosissima (gray triangle) werealigned with putative orthologs of higher plants, which were obtainedfrom the protein database of National Center for BiotechnologyInformation (NCBI). The phylogenic tree was built with the MEGA6software, using the minimum-evolution method with 1000 number ofbootstrap replication.

FIG. 2 are the results of RT-PCR analysis showing spatial expression ofCuphea LPAT genes in diverse tissues. The Cuphea eIF4 and actin wereused as internal controls.

FIG. 3 are photographs showing the subcellular localization of CupheaLPATs. Single plane image of tobacco epidermal cells was obtained fromconfocal laser scanning microscopy. Left panels are YFP signals ofCuphea LPATs, middle panels are ER markers (A, C and D) and autofluorescence of chloroplasts (B), and right panels are merged images.Bars=10 μm. (A) CpuLPATB; (B) CpuLPAT1; (C) CpuLPAT2a; (D) CvLPAT2.

FIG. 4 is data showing the complementation test of Cuphea LPATs inmutated E. coli. The new Cuphea LPATs were transformed into the E. coliJC201 strain, a mutant strain that will not grow at non-permissivetemperatures without a functional LPAT.

FIG. 5A is a graph showing the fatty acid composition of lipids (Mol %of TAG).

FIG. 5B is a graph showing the fatty acid composition of the sn-2position of seed oil TAG.

FIG. 5C is a graph showing the total amount of FAME (μg)/weight (mg).

FIG. 5D is a graph showing the fatty acid composition of lipids (Mol %of TAG).

FIG. 5E is a graph showing the fatty acid composition of the sn-2position of seed oil TAG.

FIG. 5F is a graph showing the total amount of FAME (μg)/weight (mg).

FIG. 6 shows data demonstrating C14:0-containing TAG species detected byNeutral Loss ESI-MS/MS. Electrospray mass spectroscopy of TAG isolatedfrom wild type (A) and 14:0 specific CpFatB2 (B) with CpuLPATB (C),CnLPATB (D), and CvLPAT2 (E). CN indicates the carbon number of TAG.

FIG. 7 provides the amino acid alignment of CpuLPATB homologs andinformation regarding the predicted transmembrane segments. (A) Aminoacid alignment using the CLUSTAWL algorithm was generated usingLPLAT-AGPAT-like domains of LPATB and LPAT1 homologs. Gray-dotted boxesindicate acyltransferase motifs. Circles and black triangles arecatalytic amino acids and binding site in acyltransferase motifs,respectively. (B) Predicted transmembrane region of CpuLPATB bydifferent programs; SOSUI, PSORTII, HMMTOP, and TMHMM server 2.0. Thenumbers indicate the amino acid residue of CpuLPATB. (C) A schematicshowing topological transmembrane and acyltransferse motifs of CpuLPATB.

FIG. 8 provides the amino acid alignment of CvLPAT2 homologs andinformation regarding the predicted transmembrane segments. (A) Aminoacid alignment using the CLUSTAWL algorithm was generated usingLPLAT-AGPAT-like domains of LPAT2 and LPAT3 homologs. Gray-dotted boxesindicate acyltransferase motifs. Circles and black triangles arecatalytic amino acids and binding site in acyltransferase motifs,respectively. (B) Predicted transmembrane region of CvLPAT2 by differentprograms; SOSUI, PSORTII, HMMTOP, and TMHMM server 2.0. The numbers areindicated the amino acid residue of CpuLPATB. (C) A schematic showingthe topological transmembrane and acyltransferase motifs of CvLPAT2.

FIG. 9 are graphs showing LPAT activity expressed in Agro-infiltratedtobacco leaf.

FIG. 10 is data showing the C10:0-containing TAG species detected byNeutral Loss ESI-MS/MS. Electrospray mass spectroscopy of TAG isolatedfrom wild type (A) and 14:0 specific ChFatB2 (B) with CnLPATB (C) andCvLPAT2 (D). CN indicates the carbon number of TAG. *: contains10/10/18:1, §: contains 10/10/20:1.

Part B

FIG. 11 is an unrooted phylogram of C. pulcherrima DGAT1 (CpDGAT1) andother hypothetical and functionally characterized DGATs. The alignmentwas generated by the CLUSTAL W program and the unrooted phylogram wasconstructed by the neighbor-joining method in MEGA4 software (Tamura etal., 2007, Mol. Biol. Evol., 24:1596-9).

FIG. 12 is an alignment of deduced amino acid sequence of CpDGAT1 withsome of its orthologs.

FIG. 13 is data showing CpDGAT1, CpDGAT2_A, CpDGAT2_B and CpDGAT2_Cexpression analysis in C. pulcherrima tissues by SQRT-PCR of cDNA fromtotal RNA. PCR products were obtained with gene specific primers forCpDGAT1, CpDGAT2_A, CpDGAT2_B or CpDGAT2_C.

FIG. 14 is a graph showing the short and medium chain fatty acid profileof CvFATb1+CpDGAT1 (T2) lines.

FIG. 15 is a graph showing the short and medium chain fatty acid profilein CvFatB1+CvLPAT2+CpDGAT1 (T2) lines.

FIG. 16 is a graph showing the fatty acid profile of TAG from transgenicCamelina plants

FIG. 17 is a graph showing the fatty acid profile of MAG speciesseparated following the digestion of TAG from mature seeds of wild typeand transgenic Camelina CvFatB1, CvFatB1+CvLPAT2, CvFatB1+CpDGAT1,CvFatB1+CvLPAT2+CpDGAT1 lines and C. pulcherrima and C. viscosissima.

FIG. 18A is a graph showing the fatty acid profile in developing seedsfrom wild type and transgenic Camelina lines expressing CvFatB1,CvFatB1+CpDGAT1, or CvFatB1+CvLPAT2+CpDGAT1 at 10 DAF (days afterflowering). At ten DAF, developing seeds contain very low amounts of10:0 (˜2.5 mol %); the main fatty acids were 16:0 (˜14 mol %), 18:1 (20mol %), 18:2 (40-44 mol %), and 18:3 (18-20 mol %). The percent share ofeach fatty acid (16:0 through 20:1) in TFA in transgenic lines wassimilar to that of wild type Camelina plants.

FIG. 18B is a graph showing the fatty acid profile in developing seedsfrom wild type and transgenic Camelina lines expressing CvFatB1,CvFatB1+CpDGAT1, or CvFatB1+CvLPAT2+CpDGAT1 at 17 DAF. 17 DAF seedscontain more medium-chain fatty acids (8:0 (4 mol %), 10:0 (up to 24 mol%), 12:0 (2.5-4 mol %), 14:0 (3 mol %)) and higher amounts of 16:0 (13mol %) in transgenic lines. In CvFatB1+CvLpat2+CpDGAT1, the amount of18:1 decreases, while 18:2, 18:3 and 20:1 are present in amounts of 15mol %, 16 mol % and 6 mol %, respectively, as compared to 21.4 mol %, 30mol % and 12.7 mol % in wild type Camelina plants.

FIG. 18C is a graph showing the fatty acid profile in developing seedsfrom wild type and transgenic Camelina lines expressing CvFatB1,CvFatB1+CpDGAT1, or CvFatB1+CvLPAT2+CpDGAT1 at 22 DAF. 22 DAF seedsproduce more medium chain fatty acids (5 mol % 8:0, 30 mol % 10:0, 7 mol% (12:0-14:0)). The amounts of 16:0, 18:0, 18:1, 18:2, 18:3 and 20:1 inCvFatB1+CvLPAT2+CpDGAT1 line are 12 mol %, 8 mol %, 12 mol %, 16 mol %,and 5 mol % as compared to 8 mol %, 13 mol %, 20 mol %, 41 mol %, and 10mol % in seeds of wild type plants. Thus, the total share of 8:0 to 16:0fatty acids in this line reaches 54 mol % of TFA as compared to 39 mol %in CvFatB1 line, 43% in CvFatB1+CpDGAT1 line and just 8 mol % in wildtype.

FIG. 18D is a graph showing the fatty acid profile in developing seedsfrom wild type and transgenic Camelina lines expressing CvFatB1,CvFatB1+CpDGAT1, or CvFatB1+CvLPAT2+CpDGAT1 at 30 DAF. In 30 DAF seedsfrom CvFatB1 line, there is 33.6 mol % of 8:0-16:0, 22.4 mol % 18:1,13.7 mol % 18:2, 16.0 mol % 18:3 and 6.6 mol % 20:1. CvFatB1+CpDGAT1transgenic lines accumulate more 10:0, and 8:0-16:0 total fatty acidamount is 37 mol % while amounts of 18:1, 18:2, 18:3 and 20:1 aresimilar to what is found in seeds from CvFatB1 line. InCvFatB1+CvLPAT2+CpDGAT1 lines, the average share of 8:0-16:0 fatty acidsis 43 mol % of TFA, 18.5 mol % being 10:0 and 13.2 mol % 18:1, 12.8 mol% 18:2, 18.3 mol % 18:3, 6.3 mol % 20:1.

FIG. 19 is a graph showing fatty acid profile of TAG from N. benthamianaleaves infiltrated with CvFatB1, CvFatB1+CpDGAT1, CvFatB1+AthDGAT1.

FIG. 20 is a graph showing DGAT activity in crude extracts of developingseeds from Wt and transgenic Camelina lines. Values are mean±SD (n=3).Results are for assays using [1-14C] 10:0-CoA, and diacylglycerol (DAG)species: 10:0/10:0 (1,2-didecanoyl-sn-glycerol) or 18:1/18:1(1,2-dioleoyl-sn-glycerol).

FIG. 21 is a graph showing the seed weight of mature seeds from Wt andtransgenic Camelina.

FIG. 22 is a graph showing the germination efficiency of transgenicCamelina seeds.

FIG. 23 is data showing 10:0/10:0 DAG containing TAG species detected byPrecursor 383.3 m/z ESI-MS/MS scans.

FIG. 24 are graphs showing fatty acid profiles of a Camelina transgenicline.

DETAILED DESCRIPTION

This disclosure is based on the discovery of novel nucleic acidsencoding acyltransferase polypeptides. Such nucleic acids, SEQ ID NOs:1, 3, 5, 7, or 9, and the polypeptides encoded thereby, SEQ ID NOs: 2,4, 6, 8, or 10, are described and characterized herein. Based on thisdiscovery, such nucleic acid sequences can be used to produce particularand unique medium-chain fatty acids (MCFAs).

As described herein, lysophosphatidic acid acyltransferase (LPAT) anddiacylglycerol acyltransferase (DGAT) catalyze sequential reactions inthe Kennedy pathway that produce triacylglycerols (TAG) in seeds andother plant tissues and organs. Triacylglycerols are the principalcomponent of vegetable oils, which are used in a variety of edibleapplications (e.g., baking, frying) as well as non-food applications,such as biofuels, lubricants, and surfactants. LPAT uses fatty acids inthe form of fatty acyl-Coenzyme A (CoA) as substrates for esterificationto the sn-2 position of lysophosphatidic acid (LPA) to form phosphatidicacid (PA). Following dephosphorylation of PA, the resultingdiacylglycerol (DAG) serves as a substrate for addition of a fatty acidin the form of fatty acyl-CoA to its sn-3 position to generatetriacylglycerol, via the activity of DGAT. LPAT activity in seeds of thetypical oilseed crops, such as canola (Brassica napus), camelina(Camelina sativa), and soybean (Glycine max) have strong specificity forunsaturated C18 fatty acid acyl-CoA substrates such as oleoyl (18:1)-,linoleoyl (18:2)-, and linolenoyl (18:3)-CoA, but little or no activitywith saturated fatty acyl-CoA substrates (Sun et al., 1988, PlantPhysiol., 88, 56-60; Oo et al., 1989, Plant Physiol., 91, 1288-1295).This activity arises predominantly from LPATs of the LPAT2 class, butalso with contributions from LPATs of the bacterial-type LPATB class[Arroyo-Caro, J. M., Chileh, T., Kazachkov, M., Zou, J., Alonso, D. L.,Garcia-Maroto, F. (2013) Plant Sci. 199-200:29-40]. The strict substratespecificity of oilseed LPATs for unsaturated fatty acyl-CoA substratesrepresents a major bottleneck for metabolic engineering of oilseeds toproduce TAG with high levels of saturated medium-chain fatty acids withC6-C14 chain-lengths for applications such as biofuels, includingbio-based Jet fuel A. These metabolic engineering strategies typicallyinvolve expression of divergent forms of the FatB acyl-ACP thioesterasethat are able to produce medium-chain fatty acids of differingchain-lengths. An LPAT from coconut of the LPATB class has beenpreviously shown to be effective at esterifying lauroyl (12:0)-CoA tothe sn-2 position of LPA to produce lauric acid-rich oils whenco-expressed with a 12:0-acyl carrier protein-specific FatB. The coconutLPATB enzyme, however, was ineffective for esterification of CoA formsof caprylic (8:0) or decanoic (10:0) acids to the LPA sn-2 position togenerate 10:0-rich TAG in an engineered oilseed (Wiberg et al., 2000,Planta, 212, 33-40). In addition, no plant LPAT2 enzymes have beenpreviously shown to have significant activity with any saturatedmedium-chain fatty acids.

DGAT enzymes occur in two forms, DGAT1 and DGAT2, based on their primarystructures. These enzymes also represent potential bottlenecks for theaccumulation of high levels of saturated medium-chain fatty acids in TAGof engineered oilseeds. DGAT2 enzymes from plants such as castor beanhave been shown to enhance the accumulation of modified fatty acids suchas ricinoleic acid. However, no specific plant DGAT1 or DGAT2 has beenpreviously been shown to be effective at promoting increasedaccumulation of saturated medium-chain fatty acids in engineeredoilseeds or to be active with DAGs rich in medium-chain fatty acids suchas decanoic acid (10:0).

The embodiment of this invention is the discovery of LPAT2 and DGAT1genes that are demonstrated in this disclosure to enhance theaccumulation of medium-chain fatty acids, in particular, C8:0 and C10:0,in TAG and in the sn-2 position of TAG when expressed together withspecialized FatB genes in seeds of the oilseed crop camelina (Camelinasativa). The co-expression of the LPAT2 genes with the DGAT1 genes isalso shown to yield synergistic increases in medium-chain fatty acidaccumulation in TAG and the sn-2 position of TAG in transgenic plants.

Nucleic Acids and Polypeptides

Novel nucleic acids encoding acyltransferases are provided herein (see,for example, SEQ ID NOs: 1, 3, 5, 7, or 9). Acyltransferases (PF01553;EC 2.3.1) are well known in the art and are defined as transferaseenzymes that act on acyl groups. The acyltransferases exemplified hereininclude a lysophosphatidic acid acyltransferase (LPAT; EC 2.3.1.51) anda diacylglycerol acyltransferase (DGAT; EC 2.3.1.20). The LPAT2 andLPAT2a polypeptides disclosed herein are unique in that they esterifysaturated C8-C16 fatty acyl-CoA, including a high affinity for saturatedC8 and C10 fatty acyl-CoA, at the sn-2 position of triacylglycerols(TAGs), while the DGAT1 polypeptides disclosed herein have uniquespecificity for diacylglycerols (DAGs) substrates having a saturated C10and, to a lesser extent, a saturated C8, at the sn-2 position.

Novel nucleic acids encoding medium-chain fatty acid (MCFA)-specificthioesterase, FatB polypeptides also are provided herein (see, forexample, SEQ ID NO: 15). FatB polypeptides are a class of thioesterases(EC 3.2.1.14) that release C8 to C16 saturated fatty acids from acylcarrier protein (ACP) during de novo fatty acid synthesis. The typicalFatB releases C16:0 from ACP, but FatBs that release other saturatedfatty acids are known.

As used herein, nucleic acids can include DNA and RNA, and includesnucleic acids that contain one or more nucleotide analogs or backbonemodifications. A nucleic acid can be single stranded or double stranded,which usually depends upon its intended use. The novel nucleic acidsprovided herein encode novel polypeptides (see, for example, SEQ ID NOs:2, 4, 6, 8, 10, or 16). Also provided are nucleic acids and polypeptidesthat differ from SEQ ID NOs: 1, 3, 5, 7, 9, or 15, and SEQ ID NOs: 2, 4,6, 8, 10, or 16, respectively. Nucleic acids and polypeptides thatdiffer in sequence from SEQ ID NOs: 1, 3, 5, 7, 9, or 15, and SEQ IDNOs: 2, 4, 6, 8, 10, or 16, can have at least 50% sequence identity(e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to SEQ ID NOs: 1, 3, 5, 7, 9, or 15, and SEQ ID NOs:2, 4, 6, 8, 10, or 16, respectively.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It alsowill be appreciated that a single sequence can align with more than oneother sequence and hence, can have different percent sequence identityvalues over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. Gaps of one or moreresidues can be inserted into a query sequence, a subject sequence, orboth, to maximize sequence alignments. For fast pairwise alignment ofnucleic acid sequences, the default parameters can be used (i.e., wordsize: 2; window size: 4; scoring method: percentage; number of topdiagonals: 4; and gap penalty: 5); for an alignment of multiple nucleicacid sequences, the following parameters can be used: gap openingpenalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.For fast pairwise alignment of polypeptide sequences, the followingparameters can be used: word size: 1; window size: 5; scoring method:percentage; number of top diagonals: 5; and gap penalty: 3. For multiplealignment of polypeptide sequences, the following parameters can beused: weight matrix: blosum; gap opening penalty: 10.0; gap extensionpenalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro,Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gappenalties: on. ClustalW can be run, for example, at the Baylor Collegeof Medicine Search Launcher website or at the European BioinformaticsInstitute website on the World Wide Web.

Changes can be introduced into a nucleic acid molecule (e.g., SEQ IDNOs: 1, 3, 5, 7, 9, or 15), thereby leading to changes in the amino acidsequence of the encoded polypeptide (e.g., SEQ ID NOs: 2, 4, 6, 8, 10,or 16). For example, changes can be introduced into nucleic acid codingsequences using mutagenesis (e.g., site-directed mutagenesis,PCR-mediated mutagenesis) or by chemically synthesizing a nucleic acidmolecule having such changes. Such nucleic acid changes can lead toconservative and/or non-conservative amino acid substitutions at one ormore amino acid residues. A “conservative amino acid substitution” isone in which one amino acid residue is replaced with a different aminoacid residue having a similar side chain (see, for example, Dayhoff etal. (1978, in Atlas of Protein Sequence and Structure, 5(Suppl.3):345-352), which provides frequency tables for amino acidsubstitutions), and a non-conservative substitution is one in which anamino acid residue is replaced with an amino acid residue that does nothave a similar side chain.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

As used herein, a “purified” polypeptide is a polypeptide that has beenseparated or purified from cellular components that naturally accompanyit. Typically, the polypeptide is considered “purified” when it is atleast 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dryweight, free from the polypeptides and naturally occurring moleculeswith which it is naturally associated. Since a polypeptide that ischemically synthesized is, by nature, separated from the components thatnaturally accompany it, a synthetic polypeptide is “purified.”

Nucleic acids can be isolated using techniques routine in the art. Forexample, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A vector or construct containing a nucleic acid (e.g., a nucleic acidthat encodes a polypeptide) also is provided. Vectors, includingexpression vectors, are commercially available or can be produced byrecombinant DNA techniques routine in the art. A vector containing anucleic acid can have expression elements operably linked to such anucleic acid, and further can include sequences such as those encoding aselectable marker (e.g., an antibiotic resistance gene). A vectorcontaining a nucleic acid can encode a chimeric or fusion polypeptide(i.e., a polypeptide operatively linked to a heterologous polypeptide,which can be at either the N-terminus or C-terminus of the polypeptide).Representative heterologous polypeptides are those that can be used inpurification of the encoded polypeptide (e.g., 6×His tag, glutathioneS-transferase (GST))

Expression elements include nucleic acid sequences that direct andregulate expression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins. Asused herein, operably linked means that a promoter or other expressionelement(s) are positioned in a vector relative to a nucleic acid in sucha way as to direct or regulate expression of the nucleic acid (e.g.,in-frame).

Vectors as described herein can be introduced into a host cell. As usedherein, “host cell” refers to the particular cell into which the nucleicacid is introduced and also includes the progeny of such a cell thatcarry the vector. A host cell can be any prokaryotic or eukaryotic cell.For example, nucleic acids can be expressed in bacterial cells such asE. coli, or in insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art. Many methods for introducing nucleicacids into host cells, both in vivo and in vitro, are well known tothose skilled in the art and include, without limitation,electroporation, calcium phosphate precipitation, polyethylene glycol(PEG) transformation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer.

Nucleic acids can be detected using any number of amplificationtechniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188) with an appropriate pair of oligonucleotides (e.g.,primers). A number of modifications to the original PCR have beendeveloped and can be used to detect a nucleic acid.

Nucleic acids also can be detected using hybridization. Hybridizationbetween nucleic acids is discussed in detail in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sections 7.37-7.57,9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclosessuitable Southern blot conditions for oligonucleotide probes less thanabout 100 nucleotides (Sections 11.45-11.46). The Tm between a sequencethat is less than 100 nucleotides in length and a second sequence can becalculated using the formula provided in Section 11.46. Sambrook et al.additionally discloses Southern blot conditions for oligonucleotideprobes greater than about 100 nucleotides (see Sections 9.47-9.54). TheTm between a sequence greater than 100 nucleotides in length and asecond sequence can be calculated using the formula provided in Sections9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2×SSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane.

A nucleic acid molecule is deemed to hybridize to a nucleic acid but notto another nucleic acid if hybridization to a nucleic acid is at least5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,50-fold, or 100-fold) greater than hybridization to another nucleicacid. The amount of hybridization can be quantitated directly on amembrane or from an autoradiograph using, for example, a PhosphorImageror a Densitometer (Molecular Dynamics, Sunnyvale, Calif.).

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody can be polyclonal or monoclonal. An antibody having specificbinding affinity for a polypeptide can be generated using methods wellknown in the art. The antibody can be attached to a solid support suchas a microtiter plate using methods known in the art. In the presence ofa polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex,or a polypeptide) is usually accomplished using detectable labels. Theterm “label” is intended to encompass the use of direct labels as wellas indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

Medium-Chain Fatty Acids and Methods of Making Medium-Chain Fatty Acids

The nucleic acids described herein, and the polypeptides encodedthereby, can be used to engineer a variety of useful medium-chain fattyacids and triacylglycerols that incorporate such medium-chain fattyacids. As used herein, medium-chain fatty acids refer to 6- to 14-carbonlong saturated fatty acids; specifically, caprioc (C6:0), caprylic(C8:0), capric (C10:0), lauric (C12:0), and myristic (C14:0) acids.Coconut and palm-kernel oils naturally contain high amounts ofmedium-chain fatty acids. Medium-chain triacylglycerols (MCTs) usuallycontain unsaturated 6- to 14-carbon fatty acid esters of glycerol, butthe nucleic acids described herein and the polypeptides encoded therebycan be used to produce TAGs containing predominantly C8:0, C10:0, orC12:0 fatty acid esters at the sn-2 position. As used herein,“predominantly” refers to at least 20% of the TAGs (e.g., at least 25%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% of the TAGs) having a C8:0or a C10:0 at the sn-2 position.

For example, FatBs that can generate C8 and C10 fatty acids (e.g.,ChFatB2 or CvFatB1) can be used in combination with at least one of thenovel acyltransferases described herein to strongly enhance theproduction of TAGs having a saturated C8 or C10 at the sn-2 position.Also for example, a FatB that can generate C14 and C16 fatty acids(e.g., CpFatB2) can be used in combination with at least one of thenovel acyltransferases described herein to strongly enhance theproduction of TAGs having a saturated C14 or C16 at the sn-2 position.It would be appreciated that other FatB sequences can be used incombination with at least one of the acyltransferases described hereinto engineer useful medium-chain fatty acids and the TAGs incorporatingthem.

Significantly, the LPATs and DGATs described herein have unprecedentedspecificity for C8 and C10 fatty acids. Also significantly, thecombination of one of the LPAT sequences disclosed herein and one of theDGAT sequences disclosed herein, in combination with a FatB sequence,results in a synergistic effect on the production of fatty acids and,consequently, the TAGs incorporating such fatty acids. Although notwishing to be bound by any particular mechanism, the observed synergylikely is because the LPATs described herein generate higher levels ofDAGs with saturated fatty acids at the sn-2 position, which likely isthe preferred substrate for DGATs described herein.

At least one of the acyltransferase sequences described herein can beexpressed (e.g., overexpressed) in a transgenic organism in order toproduce oils or triacylglycerols one or more medium-chain fatty acids.Therefore, transgenic organisms are provided that are transformed withat least one of the acyltransferase nucleic acid molecules describedherein (e.g., SEQ ID NOs: 1, 3, 5, 7, or 9) or a functional fragmentthereof, under control of a promoter that is able to drive expression.As discussed herein, a nucleic acid molecule used in a plant expressionvector can have a different sequence than a sequence described herein,which can be expressed as a percent sequence identity (relative to,e.g., SEQ ID NOs: 1, 3, 5, 7, or 9) or based on the conditions underwhich the sequence hybridizes to, e.g., SEQ ID NOs: 1, 3, 5, 7, or 9.

As an alternative to using a full-length sequence, a portion of thesequence can be used that encodes a polypeptide fragment having thedesired functionality (referred to herein as a “functional fragment”).When used with respect to nucleic acids, it would be appreciated that itis not the nucleic acid fragment that possesses functionality but theencoded polypeptide fragment. Based on the disclosure herein, one ofskill in the art can predict the portion(s) of a polypeptide (e.g., oneor more domains) that may impart the desired functionality.

In addition to at least one of the acyltransferases disclosed herein,the organisms also can contain a nucleic acid encoding a MCFA-specificthioesterase (FatB). Numerous FatB sequences are known in the art (e.g.,without limitation, SEQ ID NOs: 11 and 13), or the novel FatB sequencedisclosed herein (e.g., SEQ ID NO:15) can be used. In some embodiments,the FatB sequence is heterologous to the organism; in some embodiments,the FatB sequence is endogenous to the organism.

Methods of introducing one or more nucleic acids (e.g., one or moreheterologous nucleic acids, one or more transgenes) into cells,including plant cells, are known in the art and include, for example,particle bombardment, Agrobacterium-mediated transformation,microinjection, polyethylene glycol-mediated transformation (e.g., ofprotoplasts, see, for example, Yoo et al. (2007, Nature Protocols,2(7):1565-72)), liposome-mediated DNA uptake, or electroporation.Following transformation of plant cells, the transgenic cells can beregenerated into transgenic plants. As described herein, expression ofthe transgene results in an organism that produces, or exhibits anincreased amount of, medium-chain fatty acids (relative to acorresponding organism not containing or not expressing the transgene).The transgenic organisms can be screened for the amount of medium-chainfatty acids, and the medium-chain fatty acids can be obtained (e.g.,purified) from the organism.

Methods of detecting medium-chain fatty acids, and methods ofdetermining the amount of one or more medium-chain fatty acids, areknown in the art and are described herein. For example, high performanceliquid chromatography (HPLC), gas liquid chromatography (GLC), liquidchromatography (LC), and ESI-MS/MS scans can be used to detect thepresence of one or more medium-chain fatty acids and/or determine theamount of one or more medium-chain fatty acids. Lipase digestion oftriacylglycerols can also be used to establish the content ofmedium-chain fatty acids at the sn-2 position of triacylglycerols.

As used herein, an “increase” refers to an increase (e.g., astatistically significant increase) in the amount of medium-chain fattyacids or oils or triacylglycerols in plants by at least about 5% up toabout 95% (e.g., about 5% to about 10%, about 5% to about 20%, about 5%to about 50%, about 5% to about 75%, about 10% to about 25%, about 10%to about 50%, about 10% to about 90%, about 20% to about 40%, about 20%to about 60%, about 20% to about 80%, about 25% to about 75%, about 50%to about 75%, about 50% to about 85%, about 50% to about 95%, and about75% to about 95%) relative to the amount from a non-transgenic organism.As used herein, statistical significance refers to a p-value of lessthan 0.05, e.g., a p-value of less than 0.025 or a p-value of less than0.01, using an appropriate measure of statistical significance, e.g., aone-tailed two sample t-test.

When the organism is a microbe, a highly expressing or constitutivepromoter can be used to direct expression of the at least oneacyltransferase. Transgenic microbes then can be cultured or fermentedin order to obtain the medium-chain fatty acids. When the organism is aplant, it generally is desirable, although not absolutely required, touse a seed-specific promoter to direct expression of the at least oneacyltransferase. Significantly, the promoters of the acyltransferasesdescribed herein are seed-specific, and thus, can be used to directexpression of the sequences in a transgenic plant. Transgenic plantshaving increased amounts of medium-chain fatty acids, compared to theamount in a corresponding non-transgenic plant, can be selected for usein, for example, a breeding program as discussed in more detail below.

Following transformation, transgenic To plants are regenerated from thetransformed cells and those plants, or a subsequent generation of thatpopulation (e.g., T₁, T₂, T₃, etc.), can be screened for the presence ofthe at least one acyltransferase (e.g., SEQ ID NOs: 1, 3, 5, 7, or 9) orfor the phenotype (e.g., an increase in the amount of medium-chain fattyacids compared to a non-transgenic plant or a transgenic plant notexpressing the transgene). Screening for plants carrying at least oneacyltransferase can be performed using methods routine in the art (e.g.,hybridization, amplification, combinations thereof) or by evaluating thephenotype (e.g., detecting and/or determining the amount of one or moremedium-chain fatty acids in the plant (e.g., in the seed)). Generally,the presence and expression of the at least one acyltransferase (e.g.,SEQ ID NOs: 1, 3, 5, 7, or 9) results in an increase of one or moremedium-chain fatty acids in the plants (e.g., in seeds from the plants)compared to a corresponding plant (e.g., having the same varietalbackground) lacking or not expressing the at least one acyltransferase.

A plant carrying the at least one acyltransferase (e.g., SEQ ID NOs: 1,3, 5, 7, or 9) can be used in a plant breeding program to create noveland useful cultivars, lines, varieties and hybrids. Thus, in someembodiments, a T₁, T₂, T₃ or later generation plant containing the atleast one acyltransferase is crossed with a second plant, and progeny ofthe cross are identified in which the at least one acyltransferase ispresent. It will be appreciated that the second plant can be one of thespecies and varieties described herein. It will also be appreciated thatthe second plant can contain the same transgene or combination oftransgenes as the plant to which it is crossed, a different transgene,or the second plant can carry a mutation or be wild type at theendogenous locus. Additionally or alternatively, a second line canexhibit a phenotypic trait such as, for example, disease resistance,high yield, height, plant maturation, stalk size, and/or leaf number perplant.

Breeding is carried out using known procedures. DNA fingerprinting, SNPor similar technologies may be used in a marker-assisted selection (MAS)breeding program to transfer or breed the transgene(s) into other lines,varieties or cultivars, as described herein. Progeny of the cross can bescreened for the transgene(s) using methods described herein, and plantshaving the transgenes described herein (e.g., SEQ ID NOs: 1, 3, 5, 7, or9) can be selected. For example, plants in the F₂ or backcrossgenerations can be screened using a marker developed from a sequencedescribed herein or a fragment thereof, using one of the techniqueslisted herein. Seed from progeny plants also can be screened for theamount of one or more medium-chain fatty acids, and those plants havingincreased amounts, compared to a corresponding plant that lacks thetransgene, can be selected. Plants identified as possessing thetransgene and/or the expected phenotype can be backcrossed orself-pollinated to create a second population to be screened.Backcrossing or other breeding procedures can be repeated until thedesired phenotype of the recurrent parent is recovered.

Successful crosses yield F₁ plants that are fertile and that can bebackcrossed with one of the parents if desired. In some embodiments, aplant population in the F₂ generation is screened for the transgeneusing standard methods (e.g., PCR with primers based upon the nucleicacid sequences disclosed herein). Selected plants are then crossed withone of the parents and the first backcross (BC₁) generation plants areself-pollinated to produce a BC₁F₂ population that is again screened forthe transgene or the phenotype. The process of backcrossing,self-pollination, and screening is repeated, for example, at least fourtimes until the final screening produces a plant that is fertile andreasonably similar to the recurrent parent. This plant, if desired, isself-pollinated and the progeny are subsequently screened again toconfirm that the plant contains the transgene and exhibits the expectedphenotype. Breeder's seed of the selected plant can be produced usingstandard methods including, for example, field testing, confirmation ofthe presence of the transgene, and/or chemical analyses of the plant(e.g., of the seed) to determine the level of medium-chain fatty acids.

The result of a plant breeding program using the transgenic plantsdescribed herein are novel and useful cultivars, varieties, lines, andhybrids. As used herein, the term “variety” refers to a population ofplants that share constant characteristics which separate them fromother plants of the same species. A variety is often, although notalways, sold commercially. While possessing one or more distinctivetraits, a variety is further characterized by a very small overallvariation between individuals within that variety. A “pure line” varietymay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques. A “line,” as distinguished from a variety, mostoften denotes a group of plants used non-commercially, for example, inplant research. A line typically displays little overall variationbetween individuals for one or more traits of interest, although theremay be some variation between individuals for other traits.

A variety can be essentially derived from another line or variety. Asdefined by the International Convention for the Protection of NewVarieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10,1972, On Oct. 23, 1978, and on Mar. 19, 1991), a variety is “essentiallyderived” from an initial variety if: a) it is predominantly derived fromthe initial variety, or from a variety that is predominantly derivedfrom the initial variety, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of the initial variety; b) it is clearly distinguishablefrom the initial variety; and c) except for the differences which resultfrom the act of derivation, it confirms to the initial variety in theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety. Essentiallyderived varieties can be obtained, for example, by the selection of anatural or induced mutant, a somaclonal variant, a variant individualplant from the initial variety, backcrossing, or transformation.

Hybrids can be produced by preventing self-pollination of female parentplants (i.e., seed parents) of a first variety, permitting pollen frommale parent plants of a second variety to fertilize the female parentplants, and allowing F₁ hybrid seeds to form on the female plants.Self-pollination of female plants can be prevented by emasculating theflowers at an early stage of flower development. Alternatively, pollenformation can be prevented on the female parent plants using a form ofmale sterility. For example, male sterility can be produced bycytoplasmic male sterility (CMS), nuclear male sterility, genetic malesterility, molecular male sterility wherein a transgene inhibitsmicrosporogenesis and/or pollen formation, or self-incompatibility.Female parent plants containing CMS are particularly useful. Inembodiments in which the female parent plants are CMS, the male parentplants typically contain a fertility restorer gene to ensure that the F₁hybrids are fertile. In other embodiments in which the female parentsare CMS, male parents can be used that do not contain a fertilityrestorer. F₁ hybrids produced from such parents are male sterile. Malesterile hybrid seed can be interplanted with male fertile seed toprovide pollen for seed-set on the resulting male sterile plants.

Varieties, lines and cultivars described herein can be used to formsingle-cross F₁ hybrids. In such embodiments, the plants of the parentvarieties can be grown as substantially homogeneous adjoiningpopulations to facilitate natural cross-pollination from the male parentplants to the female parent plants. The F₂ seed formed on the femaleparent plants is selectively harvested by conventional means. One alsocan grow the two parent plant varieties in bulk and harvest a blend ofF₁ hybrid seed formed on the female parent and seed formed upon the maleparent as the result of self-pollination. Alternatively, three-waycrosses can be carried out wherein a single-cross F₁ hybrid is used as afemale parent and is crossed with a different male parent. As anotheralternative, double-cross hybrids can be created wherein the F₁ progenyof two different single-crosses are themselves crossed.Self-incompatibility can be used to particular advantage to preventself-pollination of female parents when forming a double-cross hybrid.

The microbial organisms used in the methods described herein include,without limitation, bacteria (E. coli, Pseudomonas sp.), cyanobacteria(Synechocystis sp), and green microalgae (C. reinhardtii, Phaeodactylumtricornutum, Chlorella sp., Nannochloropsis sp.) species, fungal(Yarrowia lipolytica, Saccharomyces cerevisiae) species. The plants usedin the methods described herein can be oilseed plants such as, withoutlimitation, Camelina spp. (e.g., Camelina sativa, Camelina alyssum,Camelina rumelica) or other Brassicaceae spp. (e.g., Brassica oleracea,Brassica rapa, Brassica napus, B. carinata), Limnanthes alba(meadowfoam), Glycine max (soybean), Linum spp. (e.g., Linumusitatissimum, flax), Crambe spp. (e.g. Crambe abyssinica), Ricinuscommunis (castor bean), Gossypium spp. (e.g. Gossypium hirsutum cotton),or non-oilseed plants such as, without limitation, legumes (e.g., peas,beans), tuberous crops (potato, cassava), or other crop plant.

The MCFAs or TAGs comprising MCFAs produced in the methods herein can beused in any number of products in which a medium-chain fatty acid or aTAG containing such a medium-chain fatty acid is desired. Such productsinclude, without limitation, biofuel and/or jet fuel. For example,vegetable oils with TAGs containing fatty acid chains having 10 or lesscarbons are more desirable feedstocks for the biofuel industry due totheir lower viscosity and because such vegetable oils may not requiretrans-esterification, which is usually a required step when convertingvegetable oils to biodiesel. In addition, the MCFAs or the TAGscomprising MCFAs produced as described herein can be used in detergents,cosmetics, surfactants, or feedstocks for preparation of otherspecialized chemicals.

In addition, in some embodiments, one or more of the acyltransferasesdescribed herein can be used in industrial inter-esterification of fattyacids to generate particular TAGs or one or more of the acyltransferasesdescribed herein can be used in a bioreactor (e.g., one or more of theacyltransferases described herein can be immobilized) to make particularTAGs for specialized nutritional or industrial applications.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Part A Example 1 Plant Material, Growth and TransformationConditions

Camelina sativa seed was sowed into 81 cm² square green plastic potswith Fafard Germination Mix based soil. Natural ambient light wassupplemented in the greenhouses with a combination of metal halide andhigh pressure sodium lights. Lights was provided for a 14 hourday-length. During the daytime temperatures were set at a range of 24°C.-26° C. and during the nighttime temperatures were set at a range 18°C.-20° C. When outdoor temperatures were above 29° C. the supplementallights were shut off to reduce the need for extra cooling. Agrobacteriumtumefaciens cells (strain C58C1) were transformed with the binaryvectors containing LPAT cDNA by the electroporation. Camelina plantswere transformed by floral dip followed by vacuum infiltration and afluorescent protein (DsRed) was used as a visual selection marker (Lu &Kang, 2008, Plant Cell Rep., 27:273-8). Segregation analyses wereperformed on the T2 showed fluorescence seeds to determine the number ofT-DNA insertion loci. Plants homozygous for the transgene wereidentified by screening T3 seeds for 100% red fluorescence.

Example 2 RNA Isolation from Cuphea Species and cDNA Conversion

Total RNAs were isolated from different Cuphea tissues such as roots,stems, leaves, flowers and developing seeds using slightly modifiedmethods described in the previous report (Chang et al., 1993, Plant Mol.Biol. Report., 11:113-6) and RNeasy Plant Mini Kit (Qiagen). The firststep was performed by the CTAB-based procedure. A pre-heated 10 ml ofextraction buffer (2% w/v CTAB, 2% w/v PVP, 2 M NaCl, 100 mM Tris-HCl pH8.0, 25 mM EDTA pH 8.0 and 0.05% w/v of spermidine) was added to thesample (200-300 mg) ground in liquid nitrogen, mixed vigorously byvortexing and incubated at 65° C. for 10 min. The sample was dividedinto several new microcentrifuge tubes. An equal volume of chloroformwas added and the tubes, mixed vigorously and then centrifuged at 13,000rpm for 10 min at 4° C. The supernatant was transferred to newmicrocentrifuge tubes and ⅓ volume of 8 M LiCl was added. The mixturewas incubated in ice for overnight, and the RNA was selectivelycollected after centrifugation at 13000 rpm for 1 hour at 4° C. Thepellet was resuspended in 500 μl of RLT buffer in RNeasy Plant Mini Kitand was then carried out as indicated in the manufacturer's handbookincluding DNase I treatment. The first-strand cDNA was synthesized from2 ug total RNA using RevertAid First Strand cDNA Synthesis Kit (ThermoScientific) with oligo-(dT) primer.

Example 3 Confocal Laser Scanning Microscope

The expression of transient fluorescent fusion proteins in tobaccoleaves was performed using the agro-infiltration methods as describedpreviously (Sparkes et al., 2006, Nature Protocols, 1:2019-25). Two daysafter infiltration, the abaxial leaf surface was observed with aconfocal laser scanning microscope (Olympus). For YFP and mCherry, theexcitation wavelengths were respectively 488 nm and 545 nm, and theemitted fluorescence was collected at 495˜530 nm and 565˜600 nm,respectively.

Example 4 Complementation of Cuphea LPAT cDNAs by Expression inEscherichia coli Mutant

The thermo-sensitive strain, E. coli JC201, was used to complement thedeficient of LPAT activity (Coleman, 1992, Mol. Gen. Genet.,232:295-303). The Cuphea LPAT cDNAs were cloned into the into thepBluescript SK⁺ multicloning site using SacI and NotI. JC201 wastransformed via heat shock and was selected on ampicillin plates at 30°C. Complementing colonies were inoculated into starter cultures andgrown to an optical density at 600 nm of 0.5 at 30° C. with or without 1mM isopropylthio-β-galactoside (IPTG). Aliquots were grown in thepresence of IPTG at 30° C. and 44° C., and growth curves wereconstructed using data obtained from three individual complementationexperiments.

Example 5 Fatty Acid Analysis of Seed Oils

Fatty acid methyl esters (FAMEs) were generated by grinding 10 mg of dryseeds in 2 mL of 2.5% H2SO4 (v/v) in methanol including 900 μg of tri17:0-TAG (Nu-Chek Prep, Elysian, Minn., USA) in toluene (10 mg/mL) as aninternal standard and heated for 45 min at 90° C. in tightly cappedtubes. Following cooling, 1.5 mL of water and 1.5 mL hexane were addedto tubes and mixed vigorously. The organic phase was transferred toautosampler vials and analyzed on an Agilent Technologies 7890A gaschromatograph (GC) fitted with a 30 m length×0.25 mm inner diameterHP-INNOWax column (Agilent, Santa Clara, Calif., USA) using H2 carriergas. The GC was programmed for an initial temperature of 90° C. (1 minhold) followed by an increase of 30° C. min-1 to 235° C. and maintainedfor a further 5 min. Detection was achieved using flame ionization.

Example 6 Neutral Loss ESI-MS/MS Analysis

Mass spectrometry analyses were conducted using an Applied Biosystems(Foster City, Calif.) 4000 QTRAP linear ion trap quadrupole massspectrometer to characterize TAG molecular species. The total neutrallipid extract for ESI-MS/MS analysis was prepared as described for seedoil content measurement below but without added internal standard anddiluted 1:5000 in water/isopropyl alcohol/methanol (55:35:10 v/v/v)containing 25 mm ammonium formate and 4 μL/L formic acid and directlyinfused into the mass spectrometer at a rate of 20 μL per minute.Instrument settings were as follows: Source temperature 400° C., ESIneedle voltage 5.5 kV (positive mode), desolvation potential (DP) 90,entrance potential (EP) 10, Curtain gas (CUR) 10, and gas 1 (GS1) 50arbitrary units, gas 2 (GS2) 40 arbitrary units. Neutral loss spectrashowing the loss of a specific fatty acid from TAG species weregenerated by monitoring the loss of 189.1 m/z (for C10:0) and 245.1 m/z(for C14:0). Scans were taken over a mass range of 500-1475 m/z with acycle time of 3 s. Data was collected for five cycles.

Example 7 RNA Isolation from Developing Seeds and cDNA LibraryConstruction

Total RNA was isolated from Cuphea pulcherrima and Cuphea viscosleavesand developing seeds collected from greenhouse grown plants andimmediately frozen in liquid nitrogen and stored at −80° C. until use inRNA isolation. Total RNA was isolated according to a method describedpreviously (Mattheus et al., 2003, Phytochemical Analysis, 14:209-15;Suzuki et al., 2004, Biotechniques, 37:542-44). In brief, developingseeds were grounded to a fine powder in liquid nitrogen. The powderswere transferred to a chilled centrifuge tube containing cold extractionbuffer consisting of 100 mM Tris-HCl, pH 8.0, 50 mM ethylenebis(oxyethylenenitrilo) tetraacetic acid, pH 8.0, 100 mM sodium chloride,1% 6-(p-toluidino)-2-naphthalenesulphonic acid, 6% sodiump-aminosalicylic acid, 1% SDS, 1% PVP-40, 3% PVPP:chloroform and 1%β-mercaptoethanol. The sample was centrifuged for 10 min with SorvallSS-34 rotor, 10500 rpm at 4° C. The supernatant was transferred to afresh tube. An equal volume of chloroform was added and the mixture wasvortexed for 2 min, centrifuged for 10 min at 10500 rpm and 4° C. Theaqueous phase was transferred to a fresh tube. The aqueous fraction wasextracted twice with phenol:chloroform (1:1, v/v), and extracted oncewith chloroform. The RNA was precipitated overnight with 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volume of 95% ethanol at −20° C. TheRNA was precipitated by centrifugation for 30 min at 10500 rpm and 4°C., rinsed once with 70% ethanol, briefly dried, and dissolved inDEPC-water.

Example 8-454 Transcriptome Analysis

200 ng of polyA+-enriched RNA prepared from developing seeds of Cupheapulcherrima and Cuphea viscosissma was used in the preparation of asingle sequencing library with custom adaptors according to methods ofNguyen, H. T., Silva, J. E., Podicheti, R., Macrander, J., Yang, W.,Nazarenus, T. J., Nam, J. W., Jaworski, J. G., Lu, C, Scheffler, B. E.,Mockaitis, K & Cahoon, E. B. (2013). The double-stranded cDNA libraryintermediate was partially normalized by DSN treatment (Evrogen) toreduce the representation of the transcripts of greatest abundance.Shearing prior to adaptor ligation was by nebulization (30 sec, 30 psi).The final library was assessed on a Bioanalyzer DNA7500 chip (Agilent)and showed a peak size of 660 bp.

Emulsion PCR and sequencing was done according to the manufacturer(Roche/454 Sequencing). Two regions of two-region plus three regions offour-region GS-FLX Titanium PicoTitre™ plate were run to 800 cycles.

The Cuphea pulcherrima and Cuphea viscosissma transcriptome assemblieswere matched by BLASTX using BLOSUM62 scoring matrix and a word size of3, to protein sequences of TAIR10 representative gene models(Arabidopsis.org on the World Wide Web) with an E-value limit of 1 e-5.The top hit(s) for each query sequence was retained based on best bitscore and E-value. Secondly TAIR10 models (above) were matched toassembly elements (isotigs and singletons) using tBLASTN with an E-valuelimit of 1 e-5. Candidate acyl lipid metabolism gene sequences wereretrieved from BLAST result sets above were trimmed to include onlythese genes. The best isotig for each isogroup was retained, trimmingout putative alternative transcripts of the same gene (as described inNguyen et al., 2013, Plant Biotechnol. J., 11(6), 759-769.

Example 9 Isolation of Multiple Putative LPAT Paralogs in Cuphea Species

To isolate specialized LPAT for medium chain acyl-CoA, we performed theRNA sequencing and assembly of over 2 million 454 sequencingpyrosequencing reads from the developing seeds of C. pulcherrima(recently re-classified as C. avigera var. pulcherrima) and C.viscosissima. Nucleotide sequence similarity to Arabidopsis LPAT2 wasused for identification of potential LPAT orthologs. Six full-lengths ofLPAT candidate genes were isolated in the 454 sequencing transcriptomeof C. pulcherrima, and one full-length of LPAT candidate gene was foundin the 454 sequencing transcriptome of C. viscosissima. We used the 7full-lengths of putative LPAT genes for further studies.

The evolutionary relationship of cuphea LPAT genes was investigatedbased on their deduced amino acid sequences to collect more informationabout relationships and to predict function for TAG accumulation. Thesequences were aligned with putative orthologs of higher plants, whichwere described by Manas-Fernandez et al. (2013, Europ. J. Lipid Sci.Technol., 115:1334-6) and Arroyo-Caro et al. (2013, Plant Sci.,199:29-40), and obtained the sequences from the protein database of theNational Center for Biotechnology Information (NCBI). LPATs have beensub-grouped by plastid LPAT (LPAT1) and microsomal LPAT, and then thelatter was further categorized into two classes, A and B. The class Amicrosomal LPATs are typical enzymes involved in synthesis of membraneglycerolipids, they show ubiquitous expression in plants and have asubstrate preference for 18:1-CoA. Based on the category of ArabidopsisLPATs, the class A microsomal LPATs were further divided into 2subgroups as LPAT2/LPAT3 group and LPAT4/LPAT5 group. The class Bmicrosomal LPAT (LPATB) is classified as a seed-specific isoform and isfound in plants accumulating unusual fatty acids in their seed oil. Eventhough LPATB is a microsomal LPAT, the class has a closer relationshipwith plastidal LPAT1 than other plant groups. However, LPATB is closerto the enzyme of other organisms, such as E. coli, yeast, and human.

Four genes of class A LPAT, one gene of LPATB, and one gene of LPAT1were found in C. pulcherrima. Based on the classification, these LPATswere named as CpuLPAT1, CpuLPAT2a, CpuLPAT2b, CpuLPAT2c, CpuLPAT3 andCpuLPATB (FIG. 1). One LPAT gene from C. viscosissima was isolated,classified as class A, and named as CvLPAT2 (FIG. 1). CpuLPATB belongedto the same group as CnLPAT, which was isolated in coconut and relatedto increase of lauric acid by incorporating this fatty acid into thesn-2 position of TAG in transgenic plants seeds (Knutzon, et al., 1999,Plant Physiol., 109:999-1006). Based on the relationship between coconutLPATB and MCFA, we assumed that CpuLPATB might involve the increase ofMCFA in TAG. So the studies were focused to reveal the function ofCpuLPATB. Because CvLPAT2 was the only LPAT gene isolated from C.viscosissima, the functional studies of CvLPAT2 were performed inparallel.

Example 10 Acyltransferase Motifs and Topology of Transmembrane Domainin Cuphea LPATs

An amino acid alignment reveals a high level of amino acid identityamong plant LPATBs (FIG. 2A). The LPLAT-AGPAT-like domain of CpuLPATBshared 82%, 79%, 77%, 74% and 70% identities with Vitis vinifera (XP002278280), Ricinus communis [AFR42414], Oryza sativa (CAH66825), Cocosnucifera [Q42670], and Limnanthes douglasii [Q42870], respectively. Thedomain showed a low identity with other organisms such as 40% (Homosapiens, NP006402), 39% (Homo sapiens, NP006403), 36% (Saccharomycescerevisiae, SLC1, P33333), and 34% (Escherichia coli, PlsC, AAA24397).We predicted that there were 4 conserved acyltransferase motifs inCpuLPATB, which are NH(X)₄D (motif I, residues 137-143), GHLRIDR (motifII, residues 178-183), FPEGTR (motif III, residues 210-215), andLPIVPIVL (motif IV, residues 237-244) (FIG. 7A). These are significantlyimportant on acyltransferase activities. Hydrophobic motif II was firstcharacterized as an acyl-CoA-binding site in animal cells and mightmodulate acyl-CoA selectivity and residue “EGT” in motif III has beenpresumed to be involved in the binding of the LPA.

To predict transmembrane domain sequences, structural analysis of thegene model-translated protein sequences was carried out in silico usingSOSUI [bp.nuap.nagoya-u.ac.jp/sosui/on the World Wide Web], PSORTII[psort.hgc.jp/form2.html on the World Wide Web], HMMTOP[enzim.hu/hmmtop/on the World Wide Web], and TMHMM server 2.0[cbs.dtu.dk/services/TMHMM/ on the World Wide Web]. All programspredicted only one transmembrane domain in CpuLPATB in a similar region(FIG. 7B). Based on the analysis of motif and transmembrane domain, thepredicted topology of CpuLPATB was presented in FIG. 7C, where allacyltransferase motifs were on the cytosolic side of the ER membrane.

The deduced amino acid sequence alignment of CvLPAT2 with CpuLPAT2a,CpuLPAT2b CpuLPAT2c, and CpuLPAT3 showed the amino acid identities as82%, 79%, 79% and 66%, respectively, in full amino acid sequences, and87%, 79%, 79% and 66%, respectively, in LPLAT-AGPAT-like domain. Theamino acid identity between CpuLPAT2b and CpuLPAT2c was the highest at97% in LPLAT-AGPAT-like domain. The key motifs in LPLAT-AGPAT-likedomains were conserved well among the LPAT2s in diverse plant species(FIG. 8A). As for Motif I [NH(X)₄D] and motif III (FPEGTR), the samesequences with LPATTB, were observed in CvLPAT2 and in LPAT2/3 groupfrom C. pulcherrima. Motif II (LPVLGW) and motif IV (NVLIPRTKGFV) wereconserved in plant LPAT2/3 group, but those sequences were completelydifferent with the LPATB's. There was a putative tyrosine phosphate sitein motif V [R(X)₆Y(X)₄A] from CvLPAT2 like the other LPAT2/3 group.Transmembrane domain of CvLAAPT2 was predicted as different numbers bydifferent programs; 4 by SOSUI, 3 by PSORTII and HMMTOP and 5 by TMHMM(FIG. 8B). All programs predicted the N-terminal located in cytosol andC-terminal located in the ER lumen. Therefore, we predicted that thereare five transmembrane domains in CvLPAT2 as seen in caster bean andpresented the predicted topology of CvLPAT2 in FIG. 8C, where motif I islocated in cytosol and motif II-IV is located in the ER lumen byseparating the third transmembrane domain.

Example 11 Tissue-Specific Expression of Cuphea LPAT Isoforms

Different LPAT isoforms showed the various expression patterns andlevels in the diverse tissues of a plant. Because LPAT has beenconsidered a narrow substrate specificity, tissue specific expression isone of the clues to presume their function. RT-PCR was performed usingcDNAs from diverse tissues, such as roots, stems, leaves, flowers anddeveloping seeds, to investigate the tissue specificity and theexpression level of LPAT genes in C. pulcherrima and C. viscosissima(FIG. 2). The transcripts of CpuLPAT1 were abundant in all testedtissues except developing seeds. CpuLPAT2b and CpuLPAT3 showedubiquitous expression patterns with a low expression levels. CpuLPAT2cwas undetectable at the same PCR cycle numbers as other LPAT genes,however, when PCR cycle numbers were increased it was slightly detectedin all tissues tested. Interestingly, CpuLPAT2a and CvLPAT2, which havevery high sequence similarity (82% in amino acid), showed the developingseed-specific expressions. CpuLPATB was also exclusively detected in thedeveloping seeds. These results indicate that CpuLPATB, CpuLPAT2a andCvLPAT2 might be involved in the accumulation of MCFAs at the sn-2position of TAG.

Example 12 CpuLPATB, CpuLPAT2a, and CvLPAT2 Localize in the ER

The ER localization of LPAT2 was confirmed in Arabidopsis and Brasiccaby immunofluorescence microscopy of tapetum cells and by immunoblottingof subcellular fraction. We investigated the subcellular localization ofCpuLPAT2a, CpuLPATB, and CvLPAT2 by using a laser scanning confocalmicroscope. Yellow fluorescence protein (YFP) was fused with C-terminalof each LPAT driven by 35S promoter. Each pro35S:LPAT:YFP wastransiently co-expressed with the ER-rk CD3-959 as the ER marker (FIG.3A-C) in tobacco leaves by agro-infiltration method. YFP signals ofCpuLPAT2a, CpuLPATB, and CvLPAT2 were detected as the reticular shapeand co-localized with ER marker. The result demonstrated that CpuLPAT2a,CpuLPATB, and CvLPAT2 are microsomal LPAT localized in the ER. We alsotested the subcellular localization of CpuLPAT1, which is classified asa plastidal form. YFP signal of CpuLPAT1 was detected in the outmembrane of chloroplasts (FIG. 3D).

Example 13 CpuLPATB Complemented the E. coli Mutant, JC201

To test the activities of Cuphea. LPATs, a complementation test wasperformed in an E. coli JC201 mutant, which is a temperature-sensitivemutant of plsC and able to grow at 30° C., but not at 42° C. Thefull-length open reading frames of Cuphea LPATs were cloned into thepBluescript SK⁺ vector. FIG. 4A showed that all Cuphea LPATs and anempty vector grew at 30° C. Occasionally the JC201 cells with an emptyvector grew at 42° C., and we increased the incubation temperature as44° C. Only JC201 containing CpuLPATB was able to grow at 44° C., butfew colonies were observed in the JC201 containing other LPATs (FIG.4A). To confirm the result, we tested their growth rate by measuring theODconcentration in process of time. As seen in FIG. 4B, the cellconcentration of JC201 increased in all tested Cuphea. LPATs and emptyvector control at 30° C. However, only CpuLPATB showed the increase ofOD concentration at 44° C. We tested the LPAT activity in the induciblevector, pET-duet, but the results were the same as above, even in thepresence or absence of IPTG, Only CpuLPATB complemented the E. colimutant JC201 a LPAT activity. This result was correlated with the aminoacid homology of LPATs between plant and E. coli. CpuLPATB shares themost similar homology with E. coli LPAT (34% in domain).

Example 14 CpuLPATB and CvLPAT2 Preferentially Incorporated 14:0 and10:0, Respectively, into the Sn-2 Position of TAG

To investigate the activities of Cuphea LPATs in planta and its utilityfor oilseed metabolic engineering, the CpuLPATB and CvLPAT2 genes wereintroduced into Camelina along with the variant FatB thioesterase genes.CpFatB2 is 14:0 specific thioesterase of Cuphea palustris (Dehesh etal., 1996, Plant Physiol., 110:203-10) and ChFatB2 is 8:0 and 10:0specific FatB thioesterase of Cuphea hookeriana (Dehesh et al., 1996,The Plant J., 9:167-72). The seed-specific glycinin promoter was used todrive the CpuLPATB and the seed-specific oleosin promoter was used todrive the CvLPAT2 for exclusive gene expression in seed. Lauricacid-specific CnLPAT was used for a comparison with CpuLPATB andCvLPAT2. The expression of CpFatB2 in Camelina showed 26.3 mol % of 14:0fatty acid. When CpFatB2 was co-experessed with CnLPAT, CpuLPATB orCvLPAT2, the levels of 14:0 fatty acid were further increased as 33.1mol %, 36.5 mol % or 32.9 mol %, respectively. The expression of ChFatB2in Camelina showed 7.4 mol % of 10:0 fatty acid. Co-expression ofChFatB2 with CnLPAT or CvLPAT2 increased the 10:0 fatty acid as 10.2 mol% or 11.8 mol %, respectively. However, CpuLPATB didn't increase the10:0 fatty acid with ChFatB2 (FIG. 5). The positional distribution ofthe MCFA was also determined in TAG. Trace amounts of 16:0 and 18:0 weredetected at the sn-2 position of TAG in wild type. The composition ofMCFA at the sn-2 position of TAG was 2.9 mol % (14:0) and 1.1 mol %(16:0) in CpFatB2 Camelina seeds. Myristic acid the sn-2 position of TAGfrom CpFatB2 Camelina seeds was significantly increased up to 14.2 mol %with CnLPAT, 19.7 mol % with CpuLPATB, and 14.8 mol % with CvLPAT2 (FIG.5B). The sn-2 position of TAG in ChFatB2 was not occupied by any MCFA orsaturated fatty acid. Co-expression of ChFatB2 with CvLPAT2 resulted inthe significant increase of 10:0 (15.4 mol %) at the sn-2 position ofTAG. However, 10:0 fatty acid was barely detected in the coexpressionline of ChFatB2 and CnLPAT. CpuLPATB didn't effect on the increase of10:0 fatty acid with ChFatB2 in the transgenic Camelina seeds. Theseresults indicate that CpuLPATB and CvLPAT2 enhance the accumulation ofthe saturated MCFAs in the TAG of Camelina seed by incorporating mediumchain acyl-CoA into the sn-2 position of LPA. CpuLPATB has a preferencetoward 14:0 fatty acid and CvLPAT2 has a preference toward myristic acidand capric acid.

Example 15 The Distribution of MCFA in TAG Molecular Species

To further investigate the metabolism of MCFA in transgenic Camelinaseeds, we performed ESI-MS analysis for the molecular species of TAGfrom Camelina producing the FatB TE. Absolute peak intensity of massspectra of TAG species from seeds expressing the FatB TE and LPAT werepresented in FIG. 6 and FIG. 10. TAG species with at least one 14:0represent in plants expressing CpFatB2 with CnLPAT, CpuLPATB, andCvLPAT2, respectively, while any MCFA was not detected in the TAG inwild-type Camelina (FIG. 6). The levels of tri-MCFA-TAG speciesincreased when CpFatB2 expressed with LPATs and the highest amounttri-MCFA-TAG species was observed in CvLPAT2. Tri-MCFA-TAG species intransgenic Camelina seeds confirmed that tested LPATs contain thepreference to saturated MCFAs and CvLPAT2 is the best FatB TE for thosesubstrates.

Part B Example 16 Cloning CpDGAT1 Sequence from C. pulcherrima

The CpDGAT1 gene sequence was identified in the C. pulcherrima 454sequence data generated as described before (Nguyen et al., 2013, PlantBiotechnol. J., 11:759-69). The ORF designated as CpDGAT1 of 1482 bpencoding 484 amino acids was PCR amplified from C. pulcherrima cDNAusing the gene specific primers.

For expression in yeast the native version of CpDGAT1 ORF was amplifiedusing the primer pair CpDGAT1BamHIf and CpDGAT1XbaIr. The ORFs for Nterminus truncated and mutated versions were generated using the forwardprimers CpDGAT1trunc_BamHI and CpDGAT1AlaBamHIf, respectively. The ORFfor the truncated version (CpDGAT1trunc) of CpDGAT1 is 70 amino acidsshorter at N terminus than the native version. The alignment showed thatthe 70 amino acid N terminus of CpDGAT1 is unique and is different fromthat of other DGAT1s while the amino acid sequence downstream the 70amino acids is highly similar to that of DGAT1s from A. thaliana, O.europea, B. napus and O. sativa. The length of the differing N terminusregion of the DGAT1s from different plant species varies. In the mutatedversion the CAT coding for ¹His was replaced by GAG coding for Ala inthe forward primer, CpDGAT1AlaBamHIf. Thus three constructspYes2_CpDGAT1, pYes2_CpDGAT1Ala, and pYes2_CpDGAT1trunc were made forexpression in yeast.

For generating plant transformation vectors the ORF encoding for CpDGAT1and CpDGAT1trunc were subcloned into NotI sites of pKMS3 vectorgenerating Glycinin promoter and terminator containing CpDGAT1 genecassette. The cassette was subsequently released by AscI to be clonedinto MluI site of pBinGlyRed3+CvFatB1 yieldingpBinGlyRed3_CvFatB1+CpDGAT1 or pBinGlyRed3_CvFatB1+CpDGAT1trunc. Thebackbone of the vector is derived from pCAMBIA0380 and was engineeredwith the DsRed marker gene under the control of theconstitutively-expressed cassava mosaic virus promoter for selection oftransgenic seeds by fluorescence (Lu and Kang, 2008, Plant Cell Rep.,27:273-8). Similarly, A. thaliana DGAT1 was subcloned into the binaryvector generating pBinGlyRed3_CvFatB1+AthDGAT1 for transformation intoCamelina.

Example 17 Phylogenetic Analysis

An unrooted phylogenetic tree of CpDGAT1 deduced amino acid sequencealong with other amino acid sequences homologous to DGAT1 or DGAT2including several functionally characterized ones was constructed. Thefunctional and phylogenetic relationships were identified by theneighbor joining program in MEGA4 (Tamura et al., 2007, Mol. Biol.Evol., 24:1596-9). The bootstrap analysis was performed with 1,000replicates.

Example 18 Yeast Transformation and Selection

The constructs pYes2_CpDGAT1, pYes2_CpDGAT1Ala, and pYes2_CpDGAT1truncwere transformed into S. cerevisiae strain H1246 (W303; MATαare1-Δ::HIS3 are2-Δ::LEU2 dga1::KanMX4 lro1-Δ::TRP1 ADE2 met ura3)(Sandager et al., 2002, J. Biol. Chem., 277:6478-82) using PEG/lithiumacetate method (Gietz et al., 1995, Yeast, 11:355-60). The yeast cellsharboring the empty pYes2 vector were used as negative control.Transformants were selected by uracil prototrophy on yeast syntheticmedium (YSM) containing 2% (w/v) glucose and lacking uracil (Invitrogen,Carlsbad, Calif. USA). For functional expression YSM containing 2% (w/v)raffinose was inoculated with the yeast transformants and grown at 28°C. for 24 h in a shaker at 350 rpm. For induction, YSM containing 2%(w/v) galactose was inoculated with raffinose-grown cultures to obtainan OD of 0.2 at 600 nm and grown at 28° C. for 48 h. For fatty acidfeeding experiments cultures were grown for 2.5 hs in YSM containing 2%galactose followed by addition of 1% (w/v) Tergitol-40 and 250 μM of theappropriate fatty acid substrate. Cells were harvested bycentrifugation, washed twice with 0.1% NaHCO3, freeze-dried and used forfatty acid, TAG analysis and microsome isolation.

Example 19 Camelina Transformation and Selection

The binary vector containing a cassette for seed specific expression ofCpDGAT1, CpDGAT1trunc or AthDGAT1 was introduced into Agrobacteriumtumefaciens by electroporation. Transgenic plants were generated byfloral dip of Camelina wt plants (Lu and Kang, 2008, Plant Cell Reports,27:273-8). Transgenic seeds among mature seeds were selected using DsRedmarker and were also PCR confirmed. Expression of transgenes indeveloping seeds was confirmed by RT-PCR.

Example 20 TAG Quantification and FA Profiling

Total lipid extraction by Bligh Dyer: 30 mg of Camelina seeds wasweighed in glass test tubes, followed by addition of 270 μl (10 mg/ml)C17-TAG and 50 μl (1 mg/ml) C17-PC. Seeds were crushed in 3 ml methanol:chloroform (2:1 v/v) by grinding with a grinder and incubated for 1 h atroom temperature with agitation. Extraction was continued by adding 1 mlof chloroform and 1.9 ml of water to a test tube and vortexed,centrifuged at 4000 rpm for 10 minutes. The organic (lower) phase wastransferred to a new test tube, 400 μl was saved fortransesterification. The rest was used for separation of TAG, DAG andPolar lipids using Supelco Supel Clean LC-Si SPE (Sigma) columns. Driedtotal lipids were redissolved in 1 ml of heptane and loaded onto LC-SiSPE columns equilibrated according to manufacturer's guidelines, oncethe sample ran through the column first wax esters were eluted with 1.5ml of 95:5 heptane: ethyl ether, second TAG fraction was eluted with 5ml of heptane: ethyl ether 80:20 (v/v). DAG was eluted with 3 mlchloroform: acetone 80:20 (v/v). Columns were washed with 6 ml ofacetone followed by elution of phospholipids with 5 mlmethanol:chloroform:water 100:50:40 (v/v/v). Total phospholipids werepooled with addition of 1.33 ml chloroform and 1.31 ml water followed byvortexing and centrifugation at 4000 rpm for 10 minutes. The organicphase containing total phospholipids was transferred into a new tube.

600 μl of polar lipid fraction was dried and transesterified the restwas redessolved in 100 μl chloroform and separated by TLC in a solventsystem consisting of CHCl3:MeOH:H2O:30% ammonium hydroxide (65:35:3:2.5v/v/v/v). Bands from the TLC plates corresponding to PC were scrapedonto wax paper and transferred to 13×100 mm test tubes.Transesterification of total lipids, TAG, and phospholipid fractions wasdone in 1 ml of 2% sulphuric acid in methanol by heating at 90° C. for30 min. Upon cooling the samples to room temperature 1 ml H₂O and 1 mlheptane was added followed by vortexing and centrifuging. Heptane layerwas transferred to GC vials and analyzed in GC.

Example 21 Isolation of C. pulcherrima DGAT1 and DGAT2 Genes

Potential genes identified as DGATs were blasted against A. thalianagene database in TAIR BLAST 2.2.8. The blast identified one gene modelhighly similar to A. thaliana DGAT1 thus named CpDGAT1. In additionthree genes two of which are similar to R. communis, V. fordii DGAT2were identified and designated as CpDGAT2_A and CpDGAT2_C, the third oneis similar to A. thaliana DGAT2 and was named CpDGAT2_B. The ORF ofCpDGAT1 is 1482 bp encoding a 484 amino acid polypeptide (Altschul etal., 1997, Nucl. Acids Res., 25:3389-402). Homology search blastanalysis of 484 deduced amino acid showed it being most identical, 59and 54%, to functionally characterized DGAT1s from A. thaliana and B.napus, respectively, while it shares ˜39% identity with mammalian DGAT1s(FIG. 11). The N terminus 78 amino acids has no sequence homology inother known homologous DGAT1s (FIG. 12), the hydrophilic N-terminus of151 and 80 residues in plants and animals, respectively, were found tobe unique for every DGAT1. Nevertheless, the rest is highly conservedand identical to DGAT1s from plant species such as A. thaliana, B.napus, R. communis and O. sativa (FIG. 12). The SOSUI secondarystructure prediction program predicted ten transmembrane regions inCpDGAT1. Similarly 8-10 hydrophobic regions were identified in DGAT1s ofdifferent origins (Liu et al., 2012, Plant Biotechnol. J., 10:862-70).The average number of residues is higher for DGAT1s than that of DGAT2scorresponding to 20 kDa difference in molecular mass. Expression ofCpDGAT1 in H1246 mutant, which contains disruptions of fouracyltransferase genes that contribute to TAG synthesis, did not storeTAG biosynthesis to the S. cerevisiae while AthDGAT1 expressing H1246yeast cells make TAG. Expression of codon optimized CpDGAT1 in H1246yeast cells did not lead to any differences. Similarly, DGAT assay usingradiolabelled 10:0 and DAG 10:0/10:0 substrates with microsomes fromCpDGAT1 expressing yeast cells did not result in formation of TAG.

Example 22 Tissue-Specific Expression of C. pulcherrima DGATs

Expression profile of CpDGAT1, CpDGAT2_A, CpDGAT2_B and CpDGAT2_C inroot, stem, leaf, flower and developing seeds of C. pulcherrima wasanalyzed (FIG. 13). The transcript abundance of the genes was normalizedto that of C. pulcherrima eukaryotic initiation factor and actin (CpeIF4and CpActin) genes. It was found that CpDGAT1 is specifically expressedin developing seeds. The three C. pulcherrima DGAT2 genes expression wasobserved in all tissues, stronger expression of CpDGAT2_A, CpDGAT2_B canbe seen in developing seeds while CpDGAT2_C is expressed at similarlevels in all tissues analyzed.

Example 23 Seed-Specific Expression of CpDGAT1 and CvLPAT2 EnhancesDecanoic Acid and Caprylic Acid Content in Camelina sativa Seeds

CpDGAT1 was expressed under seed specific glycinin-1 promoter along withthe C. viscosissima thioesterase (CvFatB1), known to be specific forC10:0, C12:0, C14:0 and C16:0 Acyl-ACP. CvLPAT2 the ORF of 1155 bp wasamplified from a cDNA prepared from total RNA from C. viscosissima fromdeveloping seeds. It was cloned into pBinGlyred vector under glycinin-1promoter.

Analysis of seeds from T2 plants of 24 independent lines expressingCvFatB1+CpDGAT1 and CvFatB1+CvLPAT2+CpDGAT1, as confirmed byreverse-transcription PCR, showed increased amounts of 10:0 (FIGS. 14and 15). The 10:0 fatty acid levels in transgenic CvFatB1+CpDGAT1 andCvFatB1+CvLPAT2+CpDGAT1 T2 seeds reached as high as 13.5 and 21.5 mol %of TFA as compared to 8.0 mol % in lines expressing only CvFatB1, whilethat of C12:0, C14:0 and C16:0 stayed similar (FIGS. 14 and 15).Significant decrease in the amounts of 18:2, 18:3 and 20:1 by 10, 18 and6 mol %, respectively, was seen in all CvFatB1+CpDGAT1 transgenic lines.The oil content in seeds from T3 homozygous transgenic lines fromCvFatB1+CpDGAT1 (FIG. 14) and CvFatB1+CvLPAT2+CpDGAT1 (FIG. 15), whichhad the highest amounts of 10:0, was not significantly affected. Inaddition to C10:0, 3 to 5 mol % of C8:0 was detected in TAG in the seedsengineered to express CvFatB1+CvLPAT2+CpDGAT1.

Example 24 Enhanced Amounts of C8:0 and C10:0 are Detected at Sn-2Position of TAG from Seeds of Transgenic Camelina Lines ExpressingCvLPAT2 or/and CpDGAT1

Stereospecific analysis of fatty acid species at sn-2 position of TAGfrom engineered seeds was conducted to assess the efficiency of assemblyof short and medium chain fatty acids in TAG. Fatty acid profile of sn-2monoacylglycerol obtained by digesting with TAG sn-1 and sn-3 specificlipase from Rhizomucor miehei (Sigma) revealed that while trace amountsof 10:0 is seen at sn-2 position of TAG from seeds of CvFatB1+CpDGAT1lines, there is a striking increase up to 20 mol % in CvFatB1+CvLPAT2line and 33.1 mol % in the relative content of 10:0 at sn-2 of TAG fromCvFatB1+CvLPAT2+CpDGAT1 line was observed (FIG. 17). In addition toC10:0, 3 to 5 mol % of C8:0 was detected in TAG sn-2 position in theseeds engineered to express CvFatB1+CvLPAT2+CpDGAT1.

The increase of 10:0 at the sn-2 position of TAG from CvFatB1 expressinglines is accompanied by reduction of 18:2 and significantly that of 18:3which is 17.4 mol % as compared to 44.3 mol % at this position in TAGfrom Wt camelina seeds. In C. pulcherrima fatty acid species at sn-2position of TAG are 8:0 (up to 97 mol %) and C10:0 (3 mol %) while in C.viscosissima it is 12.6 mol % of C8:0 and 87.4 mol % of 10:0 (FIG. 17).

Example 25 DAG Species from C. sativa Transgenic Lines OverexpressingCuphea Species Acyltransferases Contain Increased Amounts of ShorterChain Saturated Fatty Acids

Fatty acid profile of DAG species from the transgenic lines was analyzed(FIG. 18). The data showed higher amounts of C10:0 and C16:0 fatty acidsin lines expressing the Cuphea acyltransferases CvLPAT2 and CpDGAT1 inaddition to the thioesterase, CvFatB1. The seeds of CvFatB1 expressinglines contain ˜4 mol % of C10:0, ˜12 mol % of C16:0, while that ofCvFatB1+CpDGAT1 and CvFatB1+CvLPAT2 contain up to 8 mol % of C10:0, 3mol % of C14:0 and 20 mol % of C16:0. DAG species fromCvFatB1+CvLPAT2+CpDGAT1 contain highest amount of C10:0 up to 14 mol %.The increase in the amounts of short and medium chain fatty acids in DAGspecies is accompanied by substantial decrease of 18:3. DAGs fromdeveloping seeds of C. viscosissima contain up to 12, 53, 8 and 32 mol %of C8:0, C10:0, C14:0 and C16:0, respectively.

Example 26 Accumulation of Short- and Medium-Chain Fatty Acids inTransgenic Camelina Lines Starts at Midstage in Developing Seeds

Fatty acid composition of developing seeds from transgenic Camelinalines were analyzed at four stages after flowering: 10 DAF, 17 DAF,22DAF and 30 DAF. Ten day developing seeds contain very low 10:0 (˜2.5mol %), main fatty acids are 16:0 (˜14 mol %), 18:1 (20 mol %), 18:2(40-44 mol %), and 18:3 (18-20 mol %), the predominant one. The percentshare of each fatty acid (C16:0 through 20:1) in TFA in transgenic linesis similar to that of wild type Camelina plants. 17 day seeds producemore of shorter chain fatty acids 8:0 (4 mol %), 10:0 (up to 24 mol %),12:0 (2.5-4 mol %), 14:0 (3 mol %) and higher amounts of 16:0 (13 mol %)in transgenic lines. In CvFatB1+CvLpat2+CpDGAT1 the amount of 18:1decreases, while 18:2, 18:3 and 20:1 make 15 mol %, 16 mol % and 6 mol%, respectively, as compared to 21.4, 30 and 12.7 mol % in wild typeCamelina plants.

22 day seeds produce more of short chain fatty acids 5 mol % C8:0, 30mol % 10:0, 7 mol % (12:0-14:0). The amounts of 16:0, 18:0, 18:1, 18:2,18:3 and 20:1 in CvFatB1+CvLPAT2+CpDGAT1 line are 12, 8, 12, 16, and 5mol % as compared to 8, 13, 20, 41, and 10 mol % in seeds of wild typeplants. Thus the share of 8:0 through 16:0 fatty acids total amount inthis line reaches 54 mol % of TFA as compared to 39 mol % in CvFatB1line, 43% in CvFatB1+CpDGAT1 line and 8 mol % in wild type.

In 30 days in seeds from CvFatB1 line there is 33.6 mol % of 8:0-16:0,22.4 mol % 18:1, 13.7 mol % 18:2, 16.0 mol % 18:3 and 6.6 mol % 20:1.CvFatB1+CpDGAT1 transgenic lines accumulate more 10:0, and 8:0-10:0total fatty acids amount is 37.5 mol % while amounts of 18:1, 18:2, 18:3and 20:1 are similar to what is found in seeds from CvFatB1 line. InCvFatB1+CvLPAT2+CpDGAT1 line the average share of 8:0-16:0 fatty acidsis 43 mol % of TFA, 18.5 mol % being 10:0 and 13.2 mol % 18:1, 12.8 mol% 18:2, 18.3 mol % 18:3, 6.3 mol % 20:1.

As seeds develop oil content increases in both wild type and transgenicCamelina lines. Major oil accumulation started in 17 days at which oilcontent doubled 26.5% as compared to 13% of dry weight in 10 days,followed by 33.4% and 28%, after 22 and 30 days, respectively, in wildtype. In CvFatB1+CvLPAT2+CpDGAT1 lines average oil content was 13%, 26%,30% and 22.7%, as compared to thioesterase only expressing lines 11.8,23.0, 24.6, and 23.4%, in 10, 17, 22 and 30 days, respectively.

Example 27 CpDGAT1 has Preference for 10:0 Containing Substrates andDecanoyl CoA

Substrate preferences of CpDGAT1 were tested using extracts from 22 daydeveloping seeds of CvFATB1, CvFatB1+CpDGAT1, andCvFatB1+CvLPAT2+CpDGAT1 (FIG. 20). Acyl-CoA dependent DGAT activity wasexamined by measuring the incorporation of [¹⁴C] acyl-CoA into DAGacceptors 10:0/10:0 (1,2-didecanoyl-sn-glycerol) or 18:1/18:1(1,2-dioleoyl-sn-glycerol). In seed extracts of CpDGAT1 expressing linesTAG formation from 1,2-DAG 10:0/10:0 and 10:0-CoA was enhanced. DGATactivity with 1,2-DAG 10:0/10:0 and 10:0-CoA was similar 80.6±4.1 and63.6±23.4 pmol TAG/min/g protein in Wt and CvFatB1 expressing line,respectively. In CvFatB1+CpDGAT1 and CvFatB1+CvLPAT2+CpDGAT1 lines theactivity was 346±77.5 and 323±57.9 pmol TAG/min/g protein, respectively.

Example 28 Germination Efficiency of Short- and Medium-Chain Fatty AcidRich Transgenic Camelina Seeds

High levels of short and medium chain fatty acids did not affect averageseed weight observed for transgenic seeds obtained in greenhouseconditions (FIG. 20). The weight of 100 seeds from wild type Camelinawas 79 mg, 74 mg for CvFatB1, 71 and 77 mg for CvFatB1+CpDGAT1, andCvFatB1+CvLPAT2+CpDGAT1 lines, respectively.

Germination efficiency of homozygous 10:0-16:0 rich transgenic Camelinaseeds was not significantly affected by high levels of 10:0 or increasedamounts of 16:0 (FIG. 22). Up to 93% of seeds from CvFatB1 linegerminated in 10 days in greenhouse conditions, for CvFatB1+CpDGAT1 itwas 78% and 97% for CvFatB1+CvLPAT2+CpDGAT1, which contains highestamounts of short and medium chain fatty acids.

Example 29 Seed-Specific Expression of CpDGAT1 and CvLPAT2a FurtherEnhances Decanoic Acid Content in Camelina sativa Seeds

CpDGAT1 was expressed under seed specific glycinin-1 promoter along withthe C. viscosissima thioesterase (CvFatB1), known to be specific forC8:0 and C10:0 Acyl-ACP. The CpuPAT2a ORF of 1164 bp was amplified fromcDNA prepared from total RNA from C. pulcherrima developing seeds andwas sub-cloned into pKMS3 vector under glycinin-1 promoters. A cassettecomprising the glycinin-1 promoter and CpuLPAT2a gene was inserted intothe pBinGlyRed-CvFatB1+CpDGAT1 to make thepBinGlyRed-CvFatB1+CpuLPAT2a+CpDGAT1.

Analysis of seeds from T2 lines expressing CvFatB1+CvLPAT2+CpDGAT1 andT3 lines expressing CvFatB1+CpuLPAT2a+CpDGAT1 showed increased amountsof 10:0 in TAG (FIGS. 16 and 24). The 10:0 fatty acid levels intransgenic CvFatB1+CvLPAT2a+CpDGAT1 T3 seeds reached as high as 18.5 and27 mol % of TAG TFA as compared to 8.0 mol % in lines expressing onlyCvFatB1, while that of C18:1, C18:2 and C18:3 stayed similar (FIGS. 16and 24). The oil content in seeds from T3 transgenic lines fromCvFatB1+CvLPAT2a+CpDGAT1 (7,11), which had high amounts of 10:0, was notsignificantly affected (FIG. 24A). The amount of C10:0 is even higher inseeds of Camelina lines expressing CpuLPAT2a in addition to CvFatB1 andCpDGAT1 (CvFatB1+CpuLPAT2a+CpDGAT1). Fatty acid profile of sn-2monoacylglycerol obtained by digesting with TAG sn-1 and sn-3 specificlipase from Rhizomucor miehei (Sigma) indicated a significant increasein 10:0 at sn-2 position of TAG from seeds of bothCvFatB1+CvLPAT2+CpDGAT1 (FIG. 17) and CvFatB1+CpuLPAT2a+CpDGAT1 (FIG.24B) lines. It is notable also that 8:0 was detected in amounts of ˜3mol % in the total TAG and in the TAG sn-2 position in seeds engineeredto express CvFatB1+CvLPAT2CpuLPAT2a+CpDGAT1.

TABLE 1 Primers used for cloning CpDGAT1 and AthDGAT1 into yeast andCamelina expression vectorsC. pulcherrima primers used for expression in yeast and CamelinaCpDGAT1BamHIf CTAGGATCCAccATGgctCATGAGGCAGTCAG HisBamHI CpDGAT1AlaBamHIfCTAGGATCCAccATGgctGAGGCAGTCAGC BamHI CpDgat1trunc_BamH1fGTCGGATCCAccATGGCTCACCGGACTTCA BamHI CpDGAT1XbaIrATATCTAGACTAGTCGATCCTTAATCCTC XbaIr CpDGlnot1FATAgcggccgcATGCATGAGGCAGTCAG BamH1

TABLE 2 Primers used for SQRT-PCR of CpDGAT1, CpDGAT2_A, CpDGAT2_B, andCpDGAT2_C Primer sequence 5′-3′ Amplicon Gene name Primer nameForward/Reverse size (bp) CpDGAT1 SQCpDG1f CTTCAATCTCTGTATGGTCACTCTC/298 SQCpDG1r GACATCAAGGCACAATCAAATCTC CpDGAT2_A Cp1DGsqfGGAGATTCGCGAGGAGCTTAAGTAGG/ 347 Cp1DGsqr CATATGGAATGTCTCCTGCACACCACCpDGAT2_B CPDG2_2 sqF GAGCGAGATGCTGAGATTGTGTTC CT/ 308 CPDG2_2 sqRTCACTGTGCACCTCATTCACCTCTTC CpDGAT2 _C CpDG2_3hinsq_FTGGTGTGCAGGAGACATTCTACATGG/ 381 CpDG2_3xbsq_R ACTTGTGCCTTGTGTCGCTCGAATAGCpActin CpACTf TTGCTTTGGACTACGAGCAGGAGA/ 189 CpACTrTGGAGTTGTAAGTCGTCTCGTGGA CpeIF4 CpeIF4_RT_F GGTGAAGCGTGACGAACTGAC/ 140CpeIF4_RT_R ∥CTCTAGTGTTCTGGTCCATGTCTCC CpDg1trF_Not1ATAgcggccgcATGGCTCACCGGACTTCA NotI CpDg1R_Not1AATGCGGCCGCCTAGTCGATCCTTAAT NotI Primers used for expression of CpDGAT2genes in yeast Cpdg1 CTAGGATCCAccATGCGGGAGGAGACGAA BamHI Cpdg2atatctagaTCAAAGGATTCTCAGTTTGA XbaI Cpdg3 CTAGGATCCAccATGATAGGGTTCaATGABamHI Cpdg4 atatctagaTCACAAAATTCTCAGTTCGA XbaI Cpdg5ctaAAGCTTAccATGGGAGAGGAGGCGGAC HindIII Cpdg6atactcgagTTAAAGTATTCTCAGTTTGA XhoI A. thaliana DGAT1 primers used forcloning into yeast and Camelina transformation AthBamHIDGAT1FCTAGGATCCAccATGGCGATTTTGGATTC BamHI AthXbaIDGAT1RATATCTAGATCATGACATCGATCCTTTTC XbaI AthNotIf ATAgcggccgcATGGCGATTTTGGATTNotIf AthNotIr

NotIr

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

What is claimed is:
 1. A method of producing triacylglycerols (TAGs)comprising medium-chain fatty acids (MCFAs) in an organism, comprising:introducing a transgene into the organism, wherein the transgenecomprises at least one nucleic acid sequence encoding anacyltransferase, wherein the at least one acyltransferase exhibits asubstrate specificity for saturated fatty acids; thereby producing TAGscomprising MCFAs in the organism.
 2. The method of claim 1, wherein atleast 20% of the TAGs comprising MCFAs have a C8:0 or a C10:0 at sn-2position.
 3. The method of claim 1, wherein the saturated fatty acidsare selected from the group consisting of C8:0 and C10:0.
 4. The methodof claim 1, wherein the at least one acyltransferase is alysophosphatidic acid acyltransferase (LPAT) or a diacylglycerolacyltransferase (DGAT).
 5. The method of claim 1, wherein the at leastone acyltransferase is a lysophosphatidic acid acyltransferase (LPAT)and a diacylglycerol acyltransferase (DGAT).
 6. The method of claim 4,wherein the nucleic acid sequence encoding the LPAT is selected from thegroup consisting of a sequence having at least 95% sequence identity toSEQ ID NO:1 and a sequence having at least 95% sequence identity to SEQID NO:3.
 7. The method of claim 4, wherein the nucleic acid sequenceencoding the DGAT is selected from the group consisting of a sequencehaving at least 95% sequence identity to SEQ ID NO:7 and a sequencehaving at least 95% sequence identity to SEQ ID NO:9.
 8. The method ofclaim 1, wherein the nucleic acid sequence encoding the at least oneacyltransferase is selected from the group consisting of a nucleic acidsequence having at least 95% sequence identity to SEQ ID NO:1, a nucleicacid sequence having at least 95% sequence identity to SEQ ID NO:3, anucleic acid sequence having at least 95% sequence identity to SEQ IDNO:7, and a nucleic acid sequence having at least 95% sequence identityto SEQ ID NO:9.
 9. The method of claim 1, wherein the organism furthercomprises a nucleic acid sequence encoding a medium-chain fatty acid(MCFA)-specific thioesterase FatB.
 10. The method of claim 9, whereinthe nucleic acid sequence encoding the MCFA-specific thioesterase FatBis selected from the group consisting of a nucleic acid sequence havingat least 95% sequence identity to SEQ ID NO:11, a nucleic acid sequencehaving at least 95% sequence identity to SEQ ID NO:13, and a nucleicacid sequence having at least 95% sequence identity to SEQ ID NO:15. 11.The method of claim 1, wherein the organism is selected from the groupconsisting of a plant and a microbe.
 12. The method of claim 11, whereinthe plant is Camelina sativa.
 13. The method of claim 1, wherein thetransgene comprises a promoter.
 14. The method of claim 13, wherein thepromoter is a seed-specific promoter.
 15. The method of claim 11,wherein the at least one nucleic acid sequence encoding anacyltransferase is operably linked to a seed-specific promoter.
 16. Themethod of claim 15, wherein the medium-chain fatty acids are produced inthe seed.
 17. The method of claim 1, wherein the introducing step isperformed using Agrobacterium transformation, particle bombardment, orelectroporation of protoplasts.
 18. A method of producingtriacylglycerols (TAGs) comprising medium-chain fatty acids (MCFAs),comprising: providing an organism comprising a transgene, wherein thetransgene comprises at least one nucleic acid sequence encoding anacyltransferase, wherein the at least one acyltransferase exhibits asubstrate specificity for saturated fatty acids; growing the organismunder appropriate conditions; and obtaining TAGs comprising MCGAs fromthe organism.
 19. The method of claim 17, wherein the TAGs are used inbiofuel, jet fuel, detergents, and chemical feedstocks.
 20. A method ofincreasing the amount of triacylglycerols (TAGs) comprising medium-chainfatty acids (MCFAs) in the seed oil of a plant, comprising: providing aplant comprising a nucleic acid encoding a FatB polypeptide; introducinga heterologous nucleic acid molecule into the plant comprising at leastone nucleic acid sequence encoding an acyltransferase, wherein the atleast one acyltransferase exhibits a substrate specificity for saturatedfatty acids, thereby increasing the amount of TAGs comprising MCFAs inthe seed oil of the plant without significantly changing the total oilcontent in the seed.